Amplify Your Findings Agilent 7100 Capillary

with fully integrated Electrophoresis System

CE/MS

Dr. Martin Greiner

Product Manager
Low Flow CE & LC Solutions
January, 28, 2013
Overview

Why Capillary Electrophoresis complements HPLC

Setup of Agilent CE/MS system
News and updates on Agilent CE/MS
New Agilent JetStream compatible ESI-sprayer
Access to iFunnel MS
New integration of CE into Agilent MassHunter software

Application examples CE/MS

Principles of Capillary Electrophoresis

For charged substances like ions, small basic or acidic drugs and typical
biomolecules, CE is an ideal tool for analysis
Separation based on compound mobility (mass/charge) in an electrical field
High resolution separations (often > 100,000 plates)
Fast separation (few minutes)
Smallest sample volumes (nL Injections)
Less sample prep required (no stationary phase, just an open glass tube)
Orthogonal technique complementing HPLC
Low consumption of sample and aqueous buffer (green method)
Comparison of Techniques
HPLC
Separation principle
Adsorption / desorption between a stationary and a liquid mobile
phase
Advantage: Chromatography as wide spread method, many publications
Disadvantage: Expensive solvents and columns (cost of ownership)
Typical samples ( for reversed phase RP-HPLC )
Neutral or slight polar compounds, small to medium size range
Advantage: Fast UHPLC gradients, good reproducibility
Disadvantage: Ions / polar compounds often unresolved, intact proteins difficult
Detection
Various detectors ranging from UV, Fluorescence, Light scattering,
Conductivity or a whole suite of MS instruments and methods
Advantage: Choice of principle to use, covering a wide range in sensitivity
Disadvantage: Not generally compatible with each method/technique
Flow rate
Pump driven constant flow in typically L/min - ml/min range
Advantage: Stable flow, identical speed of compounds at detection point
Disadvantage: High solvent consumption, technology needs maintenance
Electrophoresis
Separation principle
Compound specific mobility in an electrical field depending on charge
state, m/z, size and shape of the molecule
Advantage: High resolution, e.g. isomers can be separated effectively
Disadvantage: Solvent and compound chemistry must be aligned
Typical samples
Ions or charged compounds, from inorganic ions to large polymers
Advantage: widest range of molecules, physiological conditions
Disadvantage: neutral compounds require often MS-incompatible additives
Detection
Main technique is UV-DAD with medium sensitivity but broad
application range. More sensitiv are LIF and Mass Spectrometry
Advantage: UV very simple to use for ppm range. MS down to ppt range
Disadvantage: Small sample volumes limits choice of detectors
Flow rate
pH dependent: capillary flow rate plus compound specific migration
Typical flow rates are in the range of x0 x00 nL/min
Advantage: Ideal for sensitive low-flow Electrospray-MS
Disadvantage: Flow rates change with chemistry (buffer capacity, wall effects)
Comparison of Techniques
HPLC Electrophoresis
Separation Columns Separation Columns
packed with adsorption material, typically RP-modified particles None, open tube fused silica capillaries usually 25 -100 m ID
Advantage: many different phases, easy switch, analytical and preparative Advantage: Low cost, low volumes of buffers (mL) reduced sample prep
Disadvantage: risk to damage packing by solvents or by matrix adsorption Disadvantage: Capillary coating might be required, non-preparative technique

Injections Injections
L-volumes (typically 1-100) liquid aliquots loaded onto the column nL-Aliquots loaded by pressure, alternative: electrokinetic injection
Advantage: increased sensitivity through high loading capacity Advantage: Stacking procedures (ITP) to concentrate compounds
Disadvantage: requires large sample volumes Disadvantage: reduced loading capacity > reduced sensitivity

UV-Detection
Light path length is an important parameter for UV-sensitivity, in HPLC
typical values are 10-60 mm in UV flow cells
Advantage: High UV sensitivity through a large light path
Disadvantage: Extra detection cell required, balance on volumes & flow rates
UV-Detection
Light path is directly through the capillary without a flow cell, this
reduces path length to the ID (25-100 m)
Advantage: Agilent bubble-cell increases ID without a loss in resolution
Disadvantage: reduced light-path > reduced sensitivity

HPLC CE
Familiarity with technique

Complementary
Analyte neutral - polar

Orthogonal

Range of detectors
Sensitivity
Reproducibility (qual/quant)
Capital cost instruments
Sample prep effort
Resolution performance
Sample volume required
Analyte size
Cost of ownership
Biocompatibility (native Prot.)
Analyte polar - fully charged
Charged polymers and Proteins
Agilent 7100 CE System

Performance Highest sensitity for UV

Handling Quick, direct and easy

Automation Agilent replenishment system

Flexibility All modes, open to external detectors

Slim housing
CE/MS Complete single vendor solution
Sensitive UV diode array detector
Economic Reducing cost of ownership based on the Agilent 1200 Series LC

Quick access cassette type

Vial sensor for sample tray

Fast Agilent 1200 electronics

Low volume replenishment system

Improved pressure/vacuum pumps

Agilent Open LAB CDS software

LabAdvisor Diagnostic tools
Agilent CE/MS Setup
OpenLAB CDS (ChemStation edition)

Sheath Liquid Pump

Single Point Software Control

7100 CE Instrument

MS Single Quadropole
CE-capillary
LC Make-Up Flow

Adapter (interface) Kit

(G1603A) Sprayer Kit
(G1607B)

Nitrogen
PC-system controlling nebulizing gas
- 7100 CE system
- MS system
- Sheath liquid pump
MS source
JetStream or ESI
On request also
Agilent CE/MS Advantages APPI / APCI

Single vendor solution: direct and competent support

Sheath Liquid Interface: robust and reliable, offering efficient control
on chemistry
Capillary outlet on ground: no compromises on voltages for CE or ESI-
MS
Agilent CE/MS Setup
New: MassHunter Software Control

Single Point Software Control

7100 CE Instrument

OF,QTOF,QQQ

LC Make-Up Flow

Agilent CE/MS Advantages

Single vendor solution:
Sheath Liquid Interface:
Capillary outlet on ground:
direct and competent support
robust and reliable, offering efficient control
on chemistry
no compromises on voltages for CE or ESI-
MS
Agilent CE/MS Solutions
Components

Agilents complete CE/MS systems consists of

Agilent Agilent 7100 CE instrument
Agilent CE/MS adapter kit (G1603A)
Agilent CE/MS sprayer Kit (G1607B)
Agilent 6000 series MS instrument portfolio (Q, QQQ, TOF, Q-TOF)
Agilent MS source (JetStream, Standard ESI, APCI, APPI)
Agilent MassHunter software B.05.01 (or higher)
Agilent HPLC pump with degasser (any 12xx-type)
Agilent HPLC pump with degasser (any 12xx-type)

Other detectors can be run in parallel to the MS-detection:

UV-DAD (included in 7100 CE)
LIF (Laser induced Fluorescence e.g. Picometrics)
CCD (contactles conductivity e.g. ISTech or eDAQ)
Agilent interface for CE/MS
Cassette type
Cassette without liquid cooling, temperature control by fast airstream providing
efficient cooling and heating using a Peltier element
Quick change of capillaries, no sealings, no liquids no leaks.
Access to 7100 built in UV-DAD providing UV monitoring (traces and full spectra)
CCD Sensors can be placed inside the temperature controlled cassette for
conductivity measurments
Airstream

MS detection
UV detection Capillary on ground

Inlet vial
High voltage
Agilent interface for CE/MS
Sheath-liquid type

Sheath liquid is added to the CE eluent by a

software controlled LC pump at a rate of typically 1 -
5 L/min.

It often consists of a mix of water, methanol or

isopropanol, adjusted for desired pH range (by
volatile acids or bases)

Besides controlling flow rate and chemical conditions

for ESI ionization of molecules it allows grounding of
the non-conductive fused silica capillary to the metal
tube of the spray needle

Advantages
High stability & reproducibility for routine analysis
Decoupling chemistry (CE separation / MS ionization)
Constant flow rates during runs and sequences
No modification of capillary / columns required
Capillary Electrophoresis
The Advantage of Agilent JetStream source compatibility
Access to latest and most sensitive MS
Saving money, no need for extra ESI sources

New setup offers:

Access to 6490 QQQ and 6550 QTOF iFunnel technology
No need to purchase separate ESI source (G1948B) for CE/MS
Full backwards compatibility of G1607B sprayer to existing ESI sources
Very robust sprayer

Old G1607A New G1607B

Sprayer tip Sprayer tip
Agilent interface for CE/MS
Agilent MassHunter software for LC-MS & CE/MS
MassHunter versions B.05.01 and higher are integrating and controlling Capillary
Electrophoresis for CE/MS analysis as a single software package under Windows 7 (64 bit)

Graphical UI

MS TIC and EIC

MS online
spectra

MS parameters
Agilent 7100 CE/MS software options

Software type OpenLAB CDS MassHunter ChemStation

(+MassHunter)
Supported CE instrument Agilent 7100 CE Agilent 7100 CE old Agilent 1600 CE

Agilent TOF,QTOF,QQQ
Supported MS instrument Agilent 61xx MSD MSD only (ChemStation)
Incl. iFunnel Systems
TOF,QTOF,QQQ (CS+MH)

Workstation Single PC Single PC 1 PC co-excecution CS + MH

1 PC-CS + 1 PC- MH

Last CS: B.04.03

SW Version C.01.03 and higher B.05.01. and higher
MH: B.04.xx

Macro (sequence >

Additional requirements
worklists)
CE/MS Applications

CE/MS analysis provides specific advantages

Orthogonal separation method vs. chromatographic separations
Very high resolution by combining CE and MS resolution
Improved MS sensitivity vs. standard UV analysis
Fast analysis especially with TOF/QTOF instrumentation
CE separation with reduced sample preparation
Non-denaturing separations of bio molecules (e.g. Proteins)

Screening Target Analysis

MS choice: TOF/QTOF MS choice: MSD/QQQ

Protein ID
Peptide mapping
Metabolomics
Small Molecule ID
Impurity analysis
.
Applications: Small anionic molecules
Metabolomics by CE-TOF/MS
Instrument type: Agilent TOF

Journal: Analytical Chemistry

 RHPEYAVSVLLR
FKDLGEEHFK
VHKECCHGDLLECADDRADLAK
LKECCDKPLLEK
SHCIAEVEK
KQTALVELLK
CCTKPESER
HPEYAVSVLLR
HLVDEPQNLIK
SLHTLFGDELCK
GACLLPK
KVPQVSTPTLVEVSR
DDPHACYSTVFDKLK
SHCIAEVEKDAIPENLPPLTADFAEDKDVCK
ALKAWSVAR
YLYEIAR

MS raw data of BSA digest

QTALVELLK
AEFVEVTK
HLVDEPQNLIK
LVNELTEFAK

DDPHACYSTVFDK
TVMENFVAFVDK

YICDNQDTISSK
MPCTEDYLSLILNR

DAIPENLPPLTADFAEDKDVCK
Peptide mapping

(sequence coverage) and final Peptide map

DAFLGSFLYEYSR

LWSTQTALA
Instrument type: Agilent Q-TOF

BioConfirm Protein Analysis Peptide Matching Results

Biopharmaceutical Analysis
Glycopeptides

More details in Agilent

Pub. No. 5990-7138EN
Quantification of Drugs of abuse
Instrument type: Agilent QQQ

Combination of effective
sample stacking method
with a highly sensitive
iFunnel QQQ instrument

Data: Isabelle Kohler, University of Geneva, School of Pharmaceutical Sciences, Geneva, Switzerland
Summary

Agilent is the only sole vendor to provide a completely integrated robust and
sensitive CE/MS solution for research and for routine analysis
Full Agilent series 6000 MS portfolio available single quad, QQQ, TOF, and
QTOF
Triple-tube interface to optimize individually separation and MS ionization no
compromises
Range of ion sources available - standard ESI and Agilent JetStream (APPI and
APCI on demand)
Flexibility on additional detectors UV-DAD, LIF, and CCD in parallel to MS
iFunnel-Sensitivity for small molecules down to the ppt range
Agilent MassHunter software control one software, one workstation
Single-vendor solution integrated system and single-source support
More information

Integrated CE/MS for orthogonal analysis is one of a

wave of innovative Agilent technologies for ensuring
your analytical lab stays at the forefront of separation
potential.
www.agilent.com/chem/cems